Molecular epidemiology and phylogenomic analysis of Mycobacterium abscessus clinical isolates in an Asian population

Mycobacterium abscessus comprises three subspecies: M. abscessus subsp. abscessus , M. abscessus subsp. bolletii , and M. abscessus subsp. massiliense . These closely related strains are typically multi-drug-resistant and can cause difficult-to-treat infections. Dominant clusters of isolates with increased pathogenic potential have been demonstrated in pulmonary infections in the global cystic fibrosis (CF) population. An investigation was performed on isolates cultured from an Asian, predominantly non-CF population to explore the phylogenomic relationships within our population and compare it to global M. abscessus isolates. Whole-genome-sequencing was performed on M. abscessus isolates between 2017 and 2019. Bioinformatic analysis was performed to determine multi-locus-sequence-type, to establish the phylogenetic relationships between isolates, and to identify virulence and resistance determinants in these isolates. A total of 210 isolates were included, of which 68.5 % (144/210) were respiratory samples. These isolates consisted of 140 (66.6 %) M . abscessus subsp. massiliense , 67 (31.9 %) M . abscessus subsp. abscessus, and three (1.4 %) M . abscessus subsp. bolletii . Dominant sequence-types in our population were similar to those of global CF isolates, but SNP differences in our population were comparatively wider despite the isolates being from the same geographical region. ESX (ESAT-6 secretory) cluster three appeared to occur most commonly in ST4 and ST6 M. abscessus subsp. massiliense , but other virulence factors did not demonstrate an association with isolate subspecies or sample source. We demonstrate that although similar predominant sequence-types are seen in our patient population, cross-transmission is absent. The risk of patient-to-patient transmission appears to be largely limited to the vulnerable CF population, indicating infection from environmental sources remains more common than human-to-human transmission. Resistance and virulence factors are largely consistent across the subspecies with the exception of clarithromycin susceptibility and ESX-3.


BACKGROUND
Non-tuberculous-mycobacteria (NTM) are environmental organisms that may result in human infections in vulnerable patient groups. Mycobacterium abscessus comprises three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. Inoculation of the bacteria following trauma or surgery may result in skin and soft tissue infections [1]. Respiratory infections usually occur in patients with underlying lung disease such as bronchiectasis, chronic obstructive pulmonary disease, and cystic fibrosis [1].
Treatment of infections of M. abscessus is fraught with difficulties due to significant antimicrobial resistance. Two guidelines are available for treatment of pulmonary infections with OPEN ACCESS M. abscessus: one by the British Thoracic Society, and a joint recommendation made by the American Thoracic Society (ATS), European Respiratory Society (ERS), European Society of Clinical Microbiology and Infectious Diseases (ESCMID), and the Infectious Diseases Society of America (IDSA) [2,3]. Multidrug treatment regimens are usually recommended, with macrolides (clarithromycin or azithromycin) and amikacin being some of the key drugs used. Clarithromycin susceptibility and administration of clarithromycin has a significant impact on treatment outcomes. The impact of other antibiotics on outcome is less clear. Treatment outcomes remain poor despite long periods of treatment. It should also be noted that current guidelines and clinical data is largely based on data from pulmonary M. abscessus infections [2,3]. Antimicrobials typically recommended for pulmonary infections may have different pharmacokinetic profiles for other infection sites and additional clinical data is required to guide management of extrapulmonary infections.
Unlike tuberculosis, M. abscessus (and NTM in general) are environmental organisms, and human-to-human spread is thought to be limited. However, there is increasing evidence of potential human-to-human transmission, with wholegenome-sequencing data used demonstrating closely-related M. abscesuss isolates within CF centres [4][5][6]. In conjunction with epidemiological links, these likely represent transmission between patients. CF is the most common predisposing risk factor for pulmonary M. abscessus infections in Caucasian populations [4,7]. Consequently, genomic analyses of clinical M. abscessus isolates have largely centred on isolates from CF patients [4]. Transmission is postulated to have occurred via generation of infectious aerosols by infected patients, and fomites [4]. Bryant et al. demonstrated that among global collection of M. abscessus isolates from CF patients, the majority of isolates formed three major clusters: two M. abscessus subsp. abscessus and one M. abscessus subsp. massiliense clusters [4]. Higher rates of phagocytosis and intracellular survival were demonstrated among clustered isolates when compared against unclustered isolates, suggesting higher pathogenic potential [4]. Murine models also demonstrated higher intracellular bacterial survival, higher bacterial burdens, and worse inflammation when infected with clustered isolates [4].
The pathology and virulence factors of M. abscessus is an under-studied field. Emerging evidence that intracellular virulence factors, namely the type VII secretion systems encoded by different ESX (ESAT-6 secretory) clusters, in particular ESX-3 and ESX-4 which appear to be unique to M. abscessus subsp. abscessus [8], the glycopeptidolipid (gpl) locus which encompasses a large set of lipid membrane transport proteins (MmpL-MmpS), and phospholipase C are important for pathogenesis [9].
Compared to Caucasian populations, CF is rare in Asians [10]. The findings of previous studies thus may not be applicable to Asian populations, particularly as comprehensive and systematic genomic analyses of M. abscessus from Asian populations are limited. Several questions remain unanswered such as whether there are also clustered clinical M. abscessus isolates in Asian populations, and whether there is similar pathogenic potential in these isolates. These could provide clues to further elucidate potential channels of transmission and pathogenicity in different patient populations. Wholegenome-sequencing of a collection of isolates from an Asian patient population was performed to explore the phylogenomic relationships within our population and compared to global M. abscessus isolates. Potential resistance mutations were also explored.

METHODS
Clinical M. abscessus isolates cultured between 1 January 2017 and 31 December 2019 for which susceptibility testing was performed were included in this study. The isolates were identified previously by Bruker MALDI Biotyper (Bruker, Billerica, Massachusetts, US). Susceptibility testing was performed during this period if microbiological criteria for pulmonary samples were fulfilled (>1 positive respiratory culture from the same patients, sample positive from a bronchoalveolar lavage). All non-pulmonary samples had susceptibility testing performed. Only the first sample for which susceptibility testing was performed was included from each patient.
The phenotypic susceptibility testing results of these isolates have been previously performed and reported [11]. This include routinely tested antimicrobials (RAPMYCO plate, Sensititre, Thermo Fisher, Waltham, Massachusetts US: trimethoprim-sulfamethoxazole ciprofloxacin, moxifloxacin, cefoxitin, amikacin, doxycycline, tigecycline, clarithromycin, linezolid, imipenem, minocycline, and tobramycin), and an extended antimicrobial panel using a customized antibiotic panel (SGPNUHS1 plate, Sensititre : vancomycin, oritavancin, dalbavancin, telavancin, rifabutin, eravacycline, delafloxacin, clofazimine, and bedaquiline). In brief, testing was performed as per manufacturer instructions and incubated at 30 °C (ambient conditions). Plates were read at 3-5 days' incubation based on whether sufficient growth was seen in the control wells. RAPMYCO plates were further incubated to 14 days if initial reading indicated clarithromycin susceptibility to exclude presence of inducible clarithromycin resistance.

Impact Statement
M. abscessus cultured from our Asian population were dominated by the same sequence type (ST) profiles seen in global cystic fibrosis (CF) populations. However, unlike in some CF centres cross-transmission with clonal isolates were not demonstrable. The infection control risks appear to be largely limited to the vulnerable CF population, indicating that infection from environmental sources is the most likely route rather than human-tohuman transmission in our setting.
Where available, the MIC results were interpreted based on CLSI breakpoints [12].

Whole-genome sequencing and bioinformatic analysis
Total genomic DNA was extracted from plate cultures using the QIAamp DNA Mini Kit (Hilden, Germany). Sequencing libraries were prepared using the NexteraXT kit (Illumina Inc., San Diego, CA, USA) and sequenced on the Illumina platform (HiSeq). Raw reads were trimmed using Trimmomatic v. 0.38 [13] then assembled with SPAdes version 3.9.0 [14]. Genome annotation was carried out using Prokka [15]. ABRicate using ResFinder database was used for genetic prediction of both acquired and chromosomal antibiotic resistance determinants. Multilocus sequence typing (MLST) was based on the PubMLST (https:// pubmlst. org/ mabscessus/) scheme using seven genes (argH, cya, gnd, murC, pta, purH, and rpoB) and performed using the MLST software available at https://githubcom/tseemann/mlst. Clustered isolates from a global CF M. abscessus collection (European Nucleotide Archive under project accession ERP001039) were also obtained and analysed [4]. The isolates used in our analysis are listed on Table S1 (available in the online version of this article). As Bryant et al. [4] did not employ the seven gene MLST scheme, we took the assembled genomes described in Table S1 in order to obtain the sequence types. For the detection of variants in drug-resistance associated genes (Table 1) Snippy v4.3.0 (https:// github. com/ tseemann/ snippy) was used while the presence and absence of putative virulence factors were determined using custom database coupled with ABRicate. FastTree [16] was used to generate phylogenetic tree based on core genome-SNPs obtained from alignment of the draft genomes using Snippy pipeline. The phylogenetic tree was visualized and annotated using iTOL [17].

Data availability
Raw sequence reads and assemblies all M. abscessus in this study have been submitted to GenBank under project accession number PRJNA734660.

RESULTS
Between 1 January 2017 and 31 December 2019, a total of 819 M. abscessus isolates were cultured from 506 patients. Susceptibility testing was performed on 268 isolates from 218 patients. A total of 210 non-duplicate isolates were included, of which 68.5 % (144/210) were respiratory samples (bronchoalveolar lavage, sputum, lung and tracheal aspirates) (Fig. 1). Only two isolates were from patients with cystic fibrosis.
An average sequencing depth of 150× was achieved for the genomes. Phylogenetic analysis classified 140 (66.6 %) as M.  (Fig. 1). The species identification was also supported by ANI values which were typically >99 % when compared to their species reference genome ( Table 2).
The overall susceptibility testing results have been previously reported without differentiating into the subspecies [11]. With the exception of clarithromycin (Fig. 1), the MIC results did not differ between the two predominant subspecies, M. abscessus subsp. abscessus and M. abscessus subsp. massiliense. The susceptibility profiles are summarized in Table 3 with MIC distributions presented in Fig. 2 Overall, we did not observe an association between specific STs and specimen sources. No predilection of dominant clones for pulmonary infections was seen (Fig. 1). There were four blood culture isolates which belonged to different STs (M. abscessus subsp. abscessus n=1, M. abscessus subsp. massiliense n=3). There were also ten peritoneal dialysate peritonitis isolates (M. abscessus subsp. abscessus n=5, M. abscessus subsp. massiliense n=5) which also had unique STs, indicating they were not clonally related (Fig. 1).

SNP analysis
Analysis of isolates from same patients by Bryant et al. [4] used 20 SNPs as the cut-off for 'probable' patient-patient transmission, and 38 SNPs as the cut-off for 'possible' recent transmission. Using these criteria, some of the isolates in our population may meet criteria for probable/possible transmission.  GenBank assembly accession numbers are provided for the reference genomes n, number of study isolates.
received within twelve days. As the isolates were received from different hospitals epidemiological links between these isolates were unlikely and the close relationship (SNPs <20) may not represent transmission events whether between individuals. Transmission from a single point-source may be possible but cannot be confirmed.

Distribution of virulence factors
Virulence genes ESX-3 and ESX-4, the glp locus and phospholipase C were sought in the genomes of our isolates. Complete modules of the ESX-3 system were not detected in M. abscessus subsp. abscessus genomes as determined by blast of all the loci (data not shown), and present in only Tigecycline Mab 0.5 1 n/a n/a n/a Mma 0.5 1 n/a n/a n/a Clofazimine Mab 0.25 0.25 n/a n/a n/a Mma 0.12 0.25 n/a n/a n/a Bedaquiline Mab 0.06 0.12 n/a n/a n/a Mma 0.06 0.12 n/a n/a n/a Eravacycline Mab 0.06 0.25 n/a n/a n/a Mma 0.12 0.25 n/a n/a n/a Rifabutin Mab 16 16 n/a n/a n/a Mma 16 32 n/a n/a n/a  (Fig. 1). We did not observe a correlation between the distribution of virulence genes and subspecies or isolates from particular specimen sites (Fig. 1).
Resistance to aminoglycosides is conferred by several mechanisms, including target mutation, drug modification, and reduced uptake and/or increased efflux [9].
Aminoglycosides-modifying enzymes are found in M. abscessus these include acetyltransferases -AAC(2′), phosphotransferase -APH(3″) and N-acetyltransferase Eis2 [9]. Out of the 210 genomes, phosphotransferases were detected in only two M. abscessus subsp. abscessus isolates. These were aph(3'')-Ic and aph (6)--Id, in RGM254 and RGM239, respectively. Both had amikacin MICs of 8 mg l −1 and the phosphotransferases did not appear to confer significant aminoglycoside resistance. Target site mutations of rrs and rpsL are responsible for high-level amikacin resistance in M. abscessus subsp. abscessus. Two isolates RGM25 and RGM172 had amikacin MICs of 128 mg l −1 however no mutations observed in rrs and rpsL indicating the possibility of other resistance mechanisms.
Overexpression of the eis2 and the multidrug efflux transporter gene (tap) and transcriptional regulator gene whiB7 have been demonstrated to be involved in the amikacin resistance in M. abscessus subsp. abscessus [21] although overexpression analysis was not investigated in this study.
Loci contributing to resistance in clofazimine, bedaquiline, tetracycline, and rifamycins (Table 1) were examined [22]. MAB_2299c which encode a putative TetR transcriptional regulator controls the expression of two separate two separate MmpS -MmpL efflux pumps (MAB_2300 -MAB_2301 and MAB_1135c-MAB_1134 c) [23,24] (Table 1). Point mutations or deletion in MAB_2299 c were commonly associated with clofazimine resistance as well as cross-resistance to bedaquiline. High levels of tetracycline and doxycycline resistance typically seen in M. abscessus are conferred by a monooxygenase, TetX (MAB_1496 c), whose expression is induced by the same antibiotics [25]. ADP-ribosyltransferase MAB_0591 is recognized as the major determinant of innate high-level rifamycin resistance in M. abscessus [26]. Overall, no meaningful SNPs (Table  S2) were detected in these loci and this was reflected in the antibiograms.

DISCUSSION
The proportion of subspecies observed here mirrored our previous study where M. abscessus subsp. massiliense was the dominant subspecies among M. abscessus identified in our laboratory [27].  [30,31]. In Japan, the geographical distribution and regional differences of the M. abscessus group indicated that amongst the M. abscessus subsp. abscessus, the proportions of C28 sequevar was highly variable, with some regions having 0 % and in other areas as high as 61.5 % [32]. Although presumed to have a positive impact on treatment outcomes, there is limited clinical data on the correlation of the C28 sequevar with outcomes in M. abscessus subsp. abscessus. Constitutive clarithromycin resistance was not identified in our collection of isolates and parallels our previous observation that constitutive resistance involving rrl mutations was rare and seen in 2.2 % isolates (2/90 isolates) [27]. Similarly, in another study, none of the 42 M. abscessus isolates exhibited point mutations in the rrl gene [33]. Treatment with clarithromycin may select for constitutive mutants over a prolonged treatment duration [34]. Although antibiotic consumption data was not studied here, this may reflect that development of rrl mutations are uncommon in the absence of antibiotic selection pressure. Despite belonging to the same MLST profile and being from the same geographical region, SNP differences in our population were comparatively wider in our patients. Clusters of isolates did not occur in our isolates as seen in CF patients. Investigations of isolates with <20 SNP differences did not demonstrate any clear links between these patients. The sample sources for these isolates were also quite diverse and included pulmonary and non-pulmonary samples, including one blood culture isolate. While healthcare-associated epidemiological links could not be established, a common exposure in the community cannot be excluded. Comparison of genomes with isolates from environmental sources may provide more clarity in the transmission of M. abscessus in the community.
We also explored isolates from specific invasive infections which may represent a common infection source. Isolates from patients with bacteraemia and patients with peritoneal-dialysisassociated-peritonitis were reviewed and were demonstrated to be from diverse sequence-types with no clonal infections (Fig. 1). There was only one identified infection of a cardiac device (RGM234; pacemaker infection). Again, these results do not suggest clonal M. abscessus infections in our population, and supports these infections have so far been sporadic unrelated events. However, it is important to note that common exposures and risk factors that may still predispose to these infections even when no clear links are established.
We demonstrate that M. abscessus cultured from our Asian population were dominated by the same ST profiles seen in global CF populations and that cross-transmission is absent. The infection control risks appear to be largely limited to the vulnerable CF population, indicating infection from environmental sources remains more common than human-to-human transmission. Virulence factors are largely consistent across the subspecies with the exception of clarithromycin susceptibility and ESX-3. Genomic resistance profiling also demonstrates that clarithromycin susceptibility remains the primary distinguishing phenotype between M. abscessus subsp. abscessus and M. abscessus subsp. massiliense, and wild-type susceptibility profiles of other antibiotics were similar between the two predominant subspecies.